We have isolated a number of clones believed to contain origins of DNA replication in monkey cells. Analysis of the distribution, frequency and time of replication of these sequences are published or in press. In addition, all have now been sequenced. In preliminary experiments, at least two appear to serve as origins for plasmid replication in vivo in monkey cells. In view of a recent report in Nature suggesting that enhancers may be required for efficient DNA replication, we have constructed a number of plasmids containing these presumptive origins plus the enhancer region of SV40. These plasmids have not yet been tested for replication. One of the presumptive origins that appears to function in monkey cells contains the consensus ARS sequence. We are, therefore, constructing tester plasmids to determine whether this and/or the other monkey sequences can serve as origins for plasmid replications in yeast (ARS). Another of the presumptive origins is derived from the region just upstream of the D loop of monkey mitochondrial DNA. We have, therefore, cloned this portion of monkey mitochondrial DNA and are in the process of sequencing this region. In other experiments we have constructed heteroduplex molecules between the SV40 replication origin and a molecule containing a substituted inverted repeat. These molecules exist as stable cruciform structures. Their behavior upon electrophoresis and their ability to bind the site-specific T antigen of SV40 have been studied.